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Related Concept Videos

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Related Experiment Video

Updated: Jul 10, 2026

Modified Terminal Restriction Fragment Analysis for Quantifying Telomere Length Using In-gel Hybridization
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Covalent Fragment Screening Using the Quantitative Irreversible Tethering Assay.

Charles P Brown1, Alan Armstrong2, David J Mann3

  • 1Department of Chemistry, Imperial College London, Molecular Sciences Research Hub; Department of Life Sciences, Imperial College London, South Kensington Campus.

Journal of Visualized Experiments : Jove
|March 17, 2025
PubMed
Summary
This summary is machine-generated.

Quantitative-irreversible tethering (qIT) is a new method to find covalent ligands for drug discovery. It accurately measures compound reactivity, prioritizing selective covalent inhibitors for therapeutic development.

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Last Updated: Jul 10, 2026

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Area of Science:

  • Biochemistry
  • Chemical Biology
  • Drug Discovery

Background:

  • Covalent compounds offer advantages as chemical probes and therapeutics by forming bonds with target proteins.
  • Electrophile-first ligand discovery is popular but complicated by varying compound reactivity with cysteines.
  • Quantitative-irreversible tethering (qIT) addresses this by normalizing data for intrinsic compound reactivity.

Purpose of the Study:

  • To demonstrate the application of qIT for generating quantitative data on ligand-protein interactions.
  • To prioritize hit ligands for further development in drug discovery.
  • To validate qIT as a robust method for identifying selective covalent inhibitors.

Main Methods:

  • Utilizing a fluorescence-based method (qIT) for hit identification and development.
  • Measuring reaction rates of individual compounds with a target protein.
  • Comparing compound reactivity with the target protein against reactivity with glutathione as a control for spontaneous reactions.

Main Results:

  • Successfully generated a quantitative and robust dataset for a target protein using qIT.
  • Enabled prioritization of hit ligands based on their preferential reactivity with the target protein.
  • Demonstrated qIT's effectiveness in identifying selective covalent fragments, building on previous successes.

Conclusions:

  • qIT is a powerful tool for identifying and developing selective covalent ligands.
  • The method normalizes for intrinsic compound reactivity, leading to more reliable hit prioritization.
  • qIT facilitates the discovery of targeted covalent inhibitors for therapeutic applications.